Chapter 106 Clinical Aspects of and Therapy for Hemophilia A Doreen B. Brettler, Elissa M. Kraus, and Peter H. Levine Introduction Hemophilia A, the most common of the true hemophilias, accounts for approximately 85% of cases. It is an X-linked recessive bleeding disorder attributable to decreased plasma levels of properly functioning factor VIII (also known as antihemophilic factor). Probably originally named haemorrhaphilia, the disease was referred to as hemophilia by Schönlein in the early 1800s. 1 In 1893, Wright 2 noted that blood from patients with hemophilia demonstrated a prolonged clotting time. In 1911, Addis 3 showed that the latter could be corrected in vitro by adding normal plasma. In 1947, both Brinkhous4 and Quick 5 independently suggested a role for factor VIII in the generation of plasma thromboplastic activity. That same year, Pavlovsky 6 correctly inferred the presence of multiple types of hemophilia by showing that he could correct the clotting defect in certain hemophilic plasma samples by adding plasma from certain other hemophilic donors. Lane 7 successfully transfused a patient with hemophilia in 1840, and plasma was first used as therapy in 1923 by Feisly. 8 Due to the minute quantities of factor VIII in plasma, its short half-life, and problems of circulatory volume overload, the vast majority of persons with hemophilia succumbed to hemorrhage early in life during the era of whole blood plasma therapy. In one study from Scandinavia, 9 for example, the mean age of death between the years 1950 and 1959 was 10.2 years for all persons with hemophilia. In 1964 Pool et al. 10 analyzed the content of the annoying precipitate that formed transiently during the thawing of fresh frozen plasma and found it to contain a disproportionately high amount of factor VIII. Thus, the era of cryoprecipitate therapy began, and this treatment made the first meaningful improvement in life expectancy. For example, in the same Scandinavian study, 9 the average age at death between 1960 and 1969 doubled to 20.1 years. The late 1960s saw the introduction of partially purified preparations of factor VIII, prepared by glycine precipitation of fresh plasma and later by polyethylene glycol precipitation. 11 More recently, therapeutic products highly purified from human plasma by the use of immunoaffinity chromatography have been available. MLID88264487 12 The first infusion of recombinant factor VIII was reported by White and colleagues in 1988. 13 Subsequently, >100 patients have been studied, all of whom have received recombinant factor VIII without difficulty. MLID91061845 14 Incidence Both Hemophilia A and B were studied by the National Heart, Lung, and Blood Institute in 1972. 15 Their estimate of incidence was 25 cases per 100,000 males. Other studies have suggested lower figures, and most experts believe the correct figure to be approximately 20 cases per 100,000 males (1 per 10,000 of the whole population), with factor VIII deficiency accounting for 85% of these. Factor IX deficiency explains 14%, and the remaining cases involve the rare congenital clotting factor factor XI, X, VII, or V deficiencies. There is no reason to believe that the incidence of hemophilia varies in different races or by geographic areas of the world. Among patients with hemophilia A being treated in the United States, 60% are classified as severe (i.e., have a factor VIII level of <1% of normal). Most of the remainder have moderate disease (factor VIII level >1% of normal). The number of people with mild disease is not known, as many such patients undoubtedly are mild bleeders and escape detection. Clinical Severity The frequency and severity of bleeding in hemophilia may be predicted from the factor VIII procoagulant level, assayed in comparison to a reference standard that is assumed to have factor VIII levels of 100%, corresponding to a factor VIII activity of 1.0 U/ml. The factor VIII level in normal persons ranges from 50% to 200% (0.50–2.0 U/ml). Those with factor VIII levels F1% of normal (F0.01 U/ml) have hemorrhages requiring therapy two to four times monthly on the average, although the range is large and the episodes are irregularly spaced. Such patients are classified as severe hemophiliacs. Those with factor VIII levels >5% of normal (>0.05 U/ml) are considered mild hemophiliacs and usually hemorrhage only due to trauma or surgery. Some such cases are not diagnosed until adult life. Occasional spontaneous hemarthrosis may occur in such patients, especially in joints damaged by previously undertreated post-traumatic hemorrhage. Patients whose factor VIII levels are between these two ranges are considered moderately severe, and their clinical picture falls between the two extremes. If such patients have had multiple untreated, or suboptimally treated hemarthroses with subsequent joint damage, the anatomic instability of these joints will cause frequent and severe bleeding, and the disease will therefore appear clinically more severe than the factor VIII assay would suggest. The choice of treatment for factor VIII deficiency depends on its severity. Those with severe or moderate disease are treated with factor concentrate. Patients with mild disease (factor VIII levels >10%) can receive desmopressin (deamino-D-arginine vasopressin [DDAVP]). Cryoprecipitate is also used in some centers to treat mild hemophilia A. However, since it cannot be virally inactivated, most physicians use factor concentrate in patients with mild hemophilia A who do not respond to DDAVP. Within a given hemophilia kindred, the clinical and laboratory severity of the disorder is relatively consistent. The appearance of a more severe clinical course in a family relative should raise the question of either established anatomic lesions that predispose to frequent or severe hemorrhage, or the development of an inhibitor. Clinical Manifestations The clinical hallmarks of hemophilia A are (1) lack of excessive hemorrhage from minor cuts or abrasions due to the normalcy of platelet function; (2) joint and muscle hemorrhages, which lead to the most difficult and disabling long-term sequelae; (3) easy bruising; (4) prolonged and potentially fatal postoperative hemorrhage; and (5) a panoply of social psychologic, vocational, and economic problems. For many patients treated with blood products prior to 1985, acquisition of human immunodeficiency virus (HIV) exacerbated already severe clinical problems. MLID87239713 16 Finally, slowly progressive hepatocellular disease, mainly attributable to chronic hepatitis C, remains a threat to patients. 17 Hemarthroses The joints most frequently involved (in descending order of frequency) are the knees, elbows, ankles, shoulders, hips, and wrists. Bleeding into the hand is rare and usually follows significant trauma, while the spine is rarely, if ever, involved. The first episodes of acute hemarthrosis occur in childhood, but often not until the child begins to walk. Infants and small children may develop large ecchymoses from being carried or lifted. Hemarthrosis is usually either spontaneous or associated with imperceptible trauma. The onset of hemorrhage is signaled by an "aura," consisting of vague warmth, a tingling sensation, and/or a sense of mild restlessness or anxiety; this aura may last up to 2 hours. Mild discomfort and slight limitation of joint motion occur next, followed (after 1 to several hours) by pain, joint swelling, cutaneous warmth, and eventual severe limitation of motion. Once bleeding has stopped, the blood is reabsorbed, and the joint returns to normal over several days to several weeks. When pain, swelling, and severe limitation of motion are present, the hemorrhage is far advanced and the process of synovitis begins; this may predispose the joint to further episodes of hemarthrosis and to hemophilic arthropathy 18 (Fig. 106-1). Joint hemorrhage should be treated after the earliest symptoms are noted and before any physician findings to prevent the long-term disabling sequelae. Adults may demonstrate periodic joint pain due to established hemophilic arthropathy rather than bleeding and may have considerable fibrosis of the joint capsule, thus preventing joint swelling. They may develop chronic limitation of motion, removing the value of this finding in diagnosing an acute bleed. Prophylactic correction of the hemostatic defect over several weeks or months can help the patient to differentiate acute hemarthrosis from the background of chronic symptoms and signs. An episode of bleeding into a joint predisposes to further episodes for at least two possible reasons. MLID75088474 19 First, hemarthrosis stimulates proliferation, chronic inflammation, and hypervascularity of the synovial membrane. Second, hemarthrosis is accompanied by rapid atrophy of the periarticular musculature and a subsequent compromise in joint protection exerted by these muscles (Fig. 106-2). Chronic Arthritis Recurrent hemarthrosis may lead to a self-perpetuating condition in which joint abnormalities persist in intervals between bleeding episodes. Clinically, the involved joint is chronically swollen, although painless and slightly warm. There are clinical findings of chronic synovitis, including prominent synovial bogginess with or without effusion (Fig. 106-3). Mild limitation of motion may be present, often with flexion deformity. Factor replacement does not modify these parameters. The likelihood of developing chronic arthritis is directly related to the overall severity of the underlying coagulation defect. In one study of 139 hemophiliac patients, only 42% were found to have definite and an additional 14% possible hemophilic arthritis, based on clinical and radiographic features. 20 Despite any previous history of hemarthrosis, however, no patient with a factor level >20% had hemophilic arthritis, while up to one-third with levels between 6% and 20% had possible or definite arthritis. In those patients who develop chronic arthritis, it is not clear whether the phenomenon is due to repeated subclinical minor hemorrhage or to irreversible synovial proliferation induced by major hemarthroses. However, it is this clinical state that often progresses to the severe destructive arthritis seen in advanced hemophilic arthropathy. The radiologic changes are seen in Figures 106-4 and 106-5. Nonsteroidal anti-inflammatory agents have been found to offer significant pain relief without routinely causing increased bleeding difficulties. End-Stage Hemophilic Arthropathy Long-standing end-stage hemophilic arthropathy has features in common with both degenerative joint disease and advanced rheumatoid arthritis. 18 The radiographic changes are shown in Figures 106-6 and 106-7. Clinically, the joint appears enlarged and "knobby," due to osteophytic bony overgrowth (Fig. 106-2). Synovial thickening and effusion, however, are not prominent. Range of motion is severely restricted, and fibrous, in contrast to bony, ankylosis is frequently seen. Subluxation, joint laxity, and malalignment are common. Hemarthroses, however, decrease in frequency. Septic Arthritis Although many diseases such as rheumatoid arthritis and osteoarthritis confer an increased risk of bacterial infection of a previously damaged joint, septic arthritis is a rarely reported complication of hemophilic arthritis. Pyogenic arthritis in hemophiliacs more commonly occurs in adults than in children and is usually monarticular, with a predilection for knee involvement. Compared with spontaneous hemarthrosis, septic arthritis is associated with a fever >38ºC within 12 hours of presentation, an increased peripheral leukocyte count, and articular pain that does not improve with replacement therapy. A predisposing factor other than hemophilic arthrop- athy is often identifiable, including previous arthrocentesis, arthroplasty, intravenous drug usage, or immunosuppression secondary to HIV-1 infection. Staphylococcus aureus is the most frequently identified organism. MLID86153874 21 Hematomas Small intramuscular hematomas are common and may resolve spontaneously, but large hematomas may lead to severe sequelae by way of compression of vital structures. Large hematomas may produce fever, leukocytosis, severe pain, and hyperbilirubinemia due to erythrocyte degradation. Those not adequately treated may result in fibrous organization with contractures. A large hematoma of the back and flank is seen in Figures 106-8 and 106-9. Psoas Hematoma Hematoma of the psoas muscle or in the muscles of the retroperitoneum may cause either pain in the lower quadrant of the abdomen (which mimics appendicitis) or pain referred to the groin (which is mistaken for hemarthrosis of the hip.) 22 Distension of the iliopsoas muscle causes the leg to be held in flexion at the hip, and compression of the femoral nerve causes pain on the anterior surface of the thigh. Increased pressure on the femoral nerve leads to paresthesia, hypesthesia, weakness of the quadriceps muscle, and even permanent paralysis of the thigh flexors. Other Closed-Space Hemorrhage Bleeding into the muscles of the forearm may lead to median or ulnar nerve paralysis or Volkmann's ischemic contracture of the hand. Calf lesions may lead to fixed equinovarus deformity at the ankle or to peroneal or other nerve palsies. Less common is wrist bleeding with nerve entrapment syndromes. Spontaneous or traumatic bleeding into the tongue or the muscles or soft tissues of the neck or throat may rapidly obstruct the airway, thereby requiring prompt and vigorous therapy. Hemophilic Cysts and Pseudotumors A large intramuscular hemorrhage may uncommonly result in the formation of a simple muscle cyst that clinically appears to be an encapsulated soft tissue area of swelling overlying muscle. Cyst formation in this setting is confined by the muscular fascial plane and results most likely from inadequate resorption of blood and clot. Subperiosteal or intraosseous hemorrhage, by contrast, may lead to a rare skeletal complication of hemophilia, a hemophilic psuedotumor. 22 Hemophilic pseudotumors are of two types: (1) the adult type that occurs proximally, usually in the pelvis or femur, and (2) a childhood type that occurs distal to the elbows or knees and carries a better prognosis than the adult type. Conservative early management of both muscle cysts and psuedotumors is indicated, including immobilization and factor replacement. If these lesions progress, however, surgical removal is indicated to avoid serious complications such as spontaneous rupture, fistula formation, neurologic or vascular entrapment, and fracture of adjacent bone. MLID77254733 23 Aspiration of a pseudotumor or cyst is contraindicated. An example of a pseudotumor is seen in Figure 106-10. Hematuria Two-thirds of hemophiliacs will have had at least one episode of hematuria. 24 Most urinary bleeding is painless, but mild flank pain may be present, and occasionally severe renal colic occurs, the latter often associated with a clot in the ureter or renal pelvis. Explanations of such bleeding vary from a trivial cause to significant underlying renal pathology. Hematuria is treated with increased fluid intake for several days and rest, followed by factor VIII for 2–4 days if the bleeding continues. Use of e-aminocaproic acid should be avoided because of the risk of preventing the lysis of clots that obstruct the ureter. Renal ultrasound or other studies are not done unless hematuria is chronic or recurrent or severe flank pain is present. Intracranial Bleeding Intracranial bleeding accounts for 25% of the hemorrhagic deaths in hemophiliacs. Antecedent trauma has occurred in one-half of such deaths. Bleeding may be subdural, epidural, subarachnoid, intracerebral, or (rarely) intraspinal. In one cooperative study of 2,500 hemophiliacs studied over 10 years, MLID78166239 25 71 episodes of central nervous system bleeding were documented; the mortality rate was 34%, and 47% of the survivors were left with mental retardation, seizure disorders, or motor impairment. Survivors had been treated with sufficient clotting factor concentration to raise the factor VIII level to 30%–50% of normal for E10–14 days. Current regimens suggest maintaining levels approaching 100%. Control of Pain in Hemophilia Pain is an extremely common problem for many persons with hemophilia. The two major causes are pain due to pressure from hemorrhage into joints, muscles, or other tissues and chronic arthritis pain, in which permanent changes have occurred in the anatomy of the joints. The control of pain is a major issue for people with hemophilia, and extensive time and energy should be put into training the patient in this regard. We believe that several general rules are important. Acute joint pain should always be assumed to be due to bleeding. At the earliest symptom of joint discomfort or limitation of motion, the correct therapy is correction of the clotting factor defect. Early application of infusion therapy prevents pain and long-term joint clotting factor defect. Patients who are taught the general philosophy “when in doubt, infuse” will wind up using less clotting factor concentrate in the long run than those who adopt a “wait and see” attitude. The latter group will have advanced lesions requiring frequent treatment and higher doses and will also develop chronic synovitis requiring many days or weeks of treatment. Chronic joint pain that fails to respond to infusion of factor VIII or IX may sometimes be arthritic pain. However, this determination should only be made after an attempt has been made to correct the coagulation defect for a period of days. If the pain that persists is accompanied by stiffness and is accentuated early in the day, it is more likely to be arthritic than hemorrhagic. In patients whose pain has hemorrhagic characteristics and who do not respond to correction of the coagulation factor defect, the use of nonsteroidal anti-inflammatory agents may provide considerable benefit. Aspirin must be avoided because of its prolonged antiplatelet effect. A major problem for most patients with hemophilia prior to the availability of adequate means of correcting the coagulopathy was narcotic addiction. Obviously, the non-narcotic agents should be used whenever possible when analgesics are required. More than 90% of patients treated at our center never use narcotic pain medicines and control their pain only with infusion therapy and the use of acetaminophen. Among older adolescents and adults with established hemarthrosis, the use of nonsteroidal drugs for arthritic pain is helpful, and at any given time between one-third and one-half of our patients in this age group will be using nonsteroidal anti-inflamma tory agents. Patients should be taught that literally hundreds of medications available over the counter contain aspirin. Lists of aspirin-containing compounds are available from a variety of sources including the National Hemophilia Foundation in New York. Our own general rule is that patients with hemophilia take no new medication of any sort until they have checked with the center. Occasionally the relief of chronic pain in hemophilia will require the use of alternative therapies, such as an exercise program administered by a physical therapist, biofeedback, acupuncture or acupressure, transcutaneous nerve stimulation, or hypnosis. A risk with hypnosis is that the patient may neglect to pay attention to episodes of pain, which indicate new hemorrhage for which the patient should be seeking medical intervention. In the last analysis the best way to control pain in hemophilia is to prevent it through the early and adequate application of replacement therapy and through the use of home infusion incorporated into a program of patient education and comprehensive hemophilia care. Other Sites of Hemorrhage Gastrointestinal hemorrhages, manifested by hematemesis, melena, or hematochezia, are unusual in hemophilia. They are usually caused by organic gastrointestinal lesions. Prolonged gingival oozing is common after shedding of deciduous teeth, eruption of new teeth, or instrumentation. If such oozing is sufficiently prolonged or severe enough to require therapy, several days of e-aminocaproic acid (Amicar) therapy will usually suffice. Infusion of coagulation factor is only occasionally needed. Epistaxis is not unusual in the severe hemophiliac, but it is unusual for mild hemophiliacs in the absence of a local nasal lesion. It is largely treated by local measures but may require factor infusion and cauterization. Post-traumatic hemorrhage requires a special comment. Normal platelet plug formation may initially provide good hemostasis but delayed bleeding usually follows trauma. For this reason, many centers advocate treatment after significant trauma whether or not evidence of hemorrhage is yet apparent. Therapy General Considerations The many ramifications of this lifelong expensive and crippling illness must be considered or the efficacy of treatment will be impaired and the outcome will be poor. Hemophilia societies are useful sources of paramedical support and information. Many problem areas exist, including the following: (1) interactional difficulties between hemophiliacs and the health care system 26; (2) psychological sequelae of overprotectiveness, such as the daredevil syndrome 27; (3) poor adjustment of the hemophilic child to school 28; (4) the enormous financial cost—in the range of $20,000–100,000/year for each patient depending on weight and severity; (5) important vocational problems in adults with established arthropathy 29; and finally, (6) a low level of education about hemophilia on the part of health care professionals, owing to the relative rarity of the disease and the pace of recent therapeutic advances. Principles of Replacement Therapy for Factor VIII The hemostatically effective plasma level for each coagulation factor is different and depends in part on the nature, extent, and duration of the bleeding lesion. The dose of replacement factor is calculated in units: 1 U is the activity of a given coagulation factor found in 1 ml of pooled, citrated fresh frozen human plasma. The factor must be given in sufficient quantity to allow for its clearance, metabolic half-life, and volume of distribution with the body. The half-life of factor VIII in plasma is between 8 and 12 hours, which includes an initial rapid decline in level owing to diffusion into extravascular pools. 30 The minimum hemostatic level of factor VIII for relatively mild hemorrhages is 30% (0.30 U/ml of plasma), while that for advanced joint or muscle bleeding or for other major hemorrhagic lesions is 50% (0.50 U/ml). One to several days of maintenance therapy is needed for such advanced lesions to resolve. Resolution is achieved by repeating the infusion at 24-hour intervals at approximately 75% of the original dose. For life-threatening lesions or surgery, levels of 80%–100% (0.80–1.00 U/ml) should be achieved and the factor VIII level should be kept above the 30%–50% range by means of appropriate doses of factor VIII infused at intervals of 8–12 hours. 31 This more frequent infusion regimen decreases the incidence of excessively low levels just prior to an infusion and also decreases the total amount of factor needed to maintain given in vivo minimum plasma levels. Constant infusion regimens are another option when levels need to be maintained above a set minimum. MLID89333689 32 Doses can be calculated by multiplying the recipient plasma volume in milliliters by the desired increment of factor VIII in units per milliliter. A simpler and reproducible dose calculation is that each unit of factor VIII infused per kilogram of body weight yields a 2% rise in plasma factor VIII level (i.e., 0.02 U/ml). An example of therapy for a 50-kg patient with an extensive laceration would include maintenance of a 30% factor VIII level in vivo until healing is complete. This can be accomplished by an initial infusion to the 60% level with 1,500 U (30 1 50 kg) of factor VIII, followed by 750 U every 12 hours thereafter for 7–10 days, with dose adjustments being made every few days as indicated by factor VIII assays. For patients with major or life-threatening lesions, the laboratory measurement of in vivo factor VIII activity is advisable because of variations that result from the responses of patients to such infusions. 33 Substitution of the activated partial thromboplastin time or another screening test for a formal factor VIII assay should be avoided because the results may be misleading. Antihemophilic factor may be given on a variety of schedules to keep the amount of in vivo factor VIII above a fixed level, thus converting a patient with severe deficiency to one with a mild or moderate deficiency. Prophylactic factor used on a regular basis in small children is being explored as a method of preventing later costly joint deterioration. Such a program can dramatically reduce the incidence of hemorrhages, but it can also increase the expense, the drain on plasma resources, and possibly the side effects as well. Indications for such prophylactic use include intensive physical therapy, recurrent hemorrhage in a single joint or chronic synovitis, or learning a new physical activity. Indications For Infusion Guidelines for doses of replacement therapy are variable from treater to treater, and there should be room for upward dose modification when the clinician is concerned that lesions are advanced or more threatening than usual. Below is a link to a table of typical initial doses of replacement therapy used at our hemophilia treatment center. Table Table Other Therapeutic Principles Acute hemarthrosis should be treated with early infusion therapy, which will minimize the risk of chronic synovitis or progressive arthropathy and avoid the need for excessive pain medication or for arthrocentesis. Aspiration of the joint during an acute hemorrhage should be avoided unless the swelling and pain are very severe or a septic joint is suspected. The total consumption of factor concentrate and health care costs are the same in patients so treated as in those in whom advanced hemorrhagic lesions develop, and the health of the patient is clearly improved. MLID75089203 34 Chronic hemophilic arthropathy can improve remarkably from several weeks or months of intensive physical therapy for muscle building and increased joint stability, intervals of avoiding weight bearing to allow for the regression of synovitis, and the correction of flexion contractures by wedging casts, night splints, or traction. 35 Regular prophylactic infusions of factor VIII can also be used to prevent traumatic bleeding. Chronic synovitis should be treated with intensive factor VIII replacement plus conservative orthopaedic and physical therapy measures, a program that produces a medical synovectomy in about one-half of the treated joints. 36 Surgical synovectomy has been suggested for patients with nonhealing chronic synovitis or with frequently recurring hemarthrosis and the progressive development of severe chronic arthropathy; it has also been successful in preventing long-term sequelae. 37 Surgical synovectomy is associated not only with a marked decrease in the frequency of hemorrhage in the joint, but also with some loss of joint motion, which may not be fully regained despite physical therapy. More recently, radioactive synovectomies, performed by injecting radioactive dysprosium into a joint, have been done successfully. Such a procedure can be considered for patients who are poor surgical risks. Joint replacement has been performed with excellent results for advanced hip arthropathy. MLID89209501 38 Although total knee replacements have now been performed in many patients, orthopaedists have generally restricted this procedure to patients with such severe knee pain that fusion is the only alternative (Fig. 106-11). Other joint prosthesis procedures, such as total shoulder replacements, are also successfully performed in persons with hemophilia. Dental care should begin with preventive dentistry early in life to minimize expense and subsequent morbidity. Restorative dentistry can now be performed with adequate local anesthesia, including the use of mandibular block, under coverage of factor VIII replacement. MLID78172062 39 For oral surgery, the use of fibrinolysis inhibitors such as e-aminocaproic acid or tranexamic acid markedly reduces the amount of coagulation factor replacement needed for hemostasis. MLID78172062 39 When e-aminocaproic acid is given orally at full therapeutic doses for 7–10 days, a single factor VIII infusion of 40 U/kg just prior to the oral surgery is often sufficient for normal hemostasis. If persistent, severe oozing occurs, another factor VIII infusion may be needed. Therapy for Mild Hemophilia A Patients with mild hemophilia A (factor VIII levels >5%) do not bleed spontaneously, but usually only after trauma or surgical procedures. The current treatment of choice for patients with factor VIII levels >10% is DDAVP (Stimate), a synthetic analogue of vasopressin. MLID89027065 40 Although its exact mechanism of action is not understood, it is thought to stimulate release of factor VIII from storage sites. MLID81134059 41 The routine dosage is 0.3 ug/kg in 50 ml of normal saline given intravenously over a period of 30–40 minutes. In a factor VIII-deficient patient, DDAVP will usually increase the factor VIII level threefold. 42 Thus, it may not be helpful in patients with factor VIII levels of <10%. In order to assess how an individual patient will respond to DDAVP, a staging test should be done. When the patient is not bleeding, a baseline factor VIII level is obtained and then the dose of DDAVP is administered. Thirty to 45 minutes after the infusion stops, a second factor VIII level is checked. The factor VIII level should rise at least threefold. If the levels rise to >80%, the response is adequate for major surgery. In some patients DDAVP can only be used for minor hemorrhages since the factor VIII levels do not rise sufficiently. DDAVP can also be used with factor concentrate in mild hemophiliacs to obtain high levels of factor VIII if needed. When DDAVP is used for major surgery, it should be given 1 hour before surgery and then every 12 hours. Tachyphylaxis may occur after repeated doses secondary to depletion of factor VIII from storage sites. MLID89027065 40 Thus factor VIII levels should be checked frequently after the first 2 days. If tachyphylaxis does occur, factor concentrate must be substituted. If a patient with mild hemophilia has an inadequate response to DDAVP, cryoprecipitate or factor concentrate must be used when the patient has surgery or encounters trauma. Since plasma-derived concentrates undergo effective virucidal procedures while cryoprecipitate cannot (and recombinant concentrate is now available), the use of factor concentrates is recommended. Although DDAVP is usually administered in a hospital or emergency room setting, a protocol can be adapted for home use. Equivalent results with subcutaneous DDAVP can be obtained, which would make home therapy much simpler. MLID88178538 43 Unfortunately, no subcutaneous preparation of DDAVP is available in the United States. Intranasal DDAVP in formulations concentrated enough to increase factor VIII levels as efficaciously as intravenous DDAVP is now available, making home management simpler. The common side effects of DDAVP include facial warmth and flushing during the infusion. Insignificant variations in blood pressure may be noted. Headaches may occur as late as 6–8 hours after the infusion. Abdominal cramping with diarrhea and generalized myalgias have rarely been noted. In very ill patients, fluid retention resulting in congestive heart failure has been reported. MLID85223521 44 In small children receiving large fluid volumes, seizures induced by severe hyponatremia after DDAVP infusion have recently occurred. MLID90053541 45 In the young pediatric age group serum sodium levels should be monitored and large amounts of intravenous fluid avoided if DDAVP is used, especially if repeated doses are given. Myocardial infarction temporally related to DDAVP infusion has been reported, MLID88094560 46 although whether DDAVP truly causes a hypercoagulable state is unclear. These serious side effects are very uncommon. In summary, DDAVP is the treatment of choice in persons with mild hemophilia A if they respond adequately. Surgery And Hemophilia Both elective and emergency surgery can be done in a patient with hemophilia A unless an inhibitor is present. Before surgery (1) a hematologist and diagnostic coagulation laboratory should be available; (2) the surgeon should feel comfortable handling a patient with a coagulation disorder; (3) there should be a blood bank or pharmacy capable of providing adequate amounts of the appropriate replacement material; (4) an appropriate rehabilitation team should be available for postoperative management, especially with orthopaedic surgery; and (5) no inhibitor should be present. An inhibitor level must be checked immediately prior to surgery. Surgery should be scheduled on Monday or Tuesday to allow for availability of laboratory services for factor level assays and best access to consultants. Preoperative orders should include “No IM medication” and “No ASA-containing compounds such as Darvon, Empirin or percodan.” For major surgery, the factor VIII level should be brought to the 80 - 100% range (40–50 U/kg) about 1 hour prior to surgery and then kept >30–50% for 10–14 days. The theoretical calculations should be checked every 2–3 days with factor VIII assays and the dose adjusted accordingly. Postoperatively, pain management should be aggressive, with patient-assisted narcotic delivery systems (PCA) or constant infusional narcotic dosing. For oral surgery such as impacted wisdom teeth removal, the factor level prior to surgery is raised to 100% with infusions. Postoperatively e-aminocaproic acid (1 g PO every 4 hours) for 7–10 days is given. If the dental procedure is minor, e-aminocaproic acid may be used alone. Patients with mild hemophilia may be able to utilize DDAVP. If there is a poor response to DDAVP, some physicians use cryoprecipitate. However, if DDABP cannot be used, we recommend factor concentrate since it can be treated with viricidal methods and is thus currently safer than cryoprecipitate. Complications of factor use include hepatitis C, which has largely been eliminated by the new production methods and hemolysis due to anti-A or anti-B in the concentrate preparation. If hemolysis occurs, blood loss should be replaced by type O packed red cells, and concentrates with low isoagglutinin titers should be obtained and utilized. Health Care Delivery The keystone of therapy in hemophilia is to provide the patient with access to immediate and adequate correction of the hemostatic defect at the earliest symptom suggestive of hemorrhage. For most persons with severe and moderately severe hemophilia, the achievement of this goal is through a combination of intensive education of the patient and family, plus the institution of a carefully supervised self-therapy program. Self-Therapy Program With the exception of patients with inhibitor antibodies, those who are unreliable or unstable, or children <3 years of age, most patients are candidates for home therapy. The day-to-day supervision of the patient may often be provided by a physician in the patient's immediate geographic vicinity, with subsequent regular visits to a hemophilia center for long-term evaluation and comprehensive care. At most centers, all patients and selected family members receive an individual half-day course on the pathophysiology, diagnosis, and therapy of hemophilia. The first several infusions are administered under medical supervision. If the patient or family demonstrate both proficiency in self-infusion and good grasp of basic principles, maximum independence is allowed. At some centers, all patients attend biannual comprehensive evaluation sessions as a minimum mandatory requirement for continuation in the program. At each session the patient is evaluated by a hematologist and an orthopaedic surgeon, a nurse, a medical social worker, an oral surgeon, a physical therapist, a vocational counselor, and, when indicated, a genetic counselor and/or a psychologist or psychiatrist. Table 106-1 Table 106-1 shows data collected on hemophiliacs treated in traditional outpatient hematology clinics for 1 year and then introduced into the formal comprehensive care program, which includes self-therapy. MLID84200752 47 It should be emphasized that the least important aspect of self-therapy is the teaching of venipuncture. The program achieves its dramatic results through patient education as well as the systematic application of the skills of a variety of appropriate medical personnel who can address the long-term problems of this lifelong disease. Because of the many problems arising from the introduction of HIV into the blood supply in the 1970s and early 1980s, 15 comprehensive hemophilia care now also involves dealing with the acquired immunodeficiency syndrome (AIDS) and HIV. 48 Patients are examined at more frequent intervals, prophylaxis with antiretroviral agents such as zidovudine is considered, and infectious disease consultation may be obtained. Additionally, intensive counseling sessions with the patient, the family, and a social worker are provided for discussion of issues such as transmission of HIV and the impact of this infection on the life of the patient. Complications of Therapy Lyophilized factor concentrates revolutionized the care of persons with hemophilia. Home therapy programs were instituted and thus the long-term effects of hemorrhage were decreased and days lost from work or school lessened. MLID84200752 47 Life span gradually increased until the AIDS era. 9 Lyophilized concentrate is a pooled product made from plasma of between 2,000 and 30,000 donors. Infectious complications from transfusion-transmitted viruses began to be noted in hemophiliacs in the late 1970s and subsequently became a major concern. MLID82159302 49 However, all factor concentrates currently produced are virally inactivated, and thus infectious complications have been greatly decreased. Increased purity of concentrates has also occurred over the ensuing years. Infectious Complications Hepatitis The major hepatitis viruses transmitted through plasma-derived concentrate and cryoprecipitate infusion are hepatitis B (HBV) and C (HCV). Acute HBV infection with elevated liver function tests, jaundice, and fever is uncommon in hemophiliacs, but approximately 90% of patients who were given infused concentrates before the current viral inactivation methods became available have developed antibody to HBV (HBsAb positive), indicating exposure. MLID85155884 50 A small percentage of patients, approximately 5%, have become chronic carriers (HBsAg positive). 51 Chronic carriers may be more prone to the development of symptomatic chronic liver disease or carcinoma of the liver. Delta hepatitis (HDV), a virus that requires the presence of HBV as a carrier, is also a potential risk to hemophilia patients, especially in endemic areas, since coinfection with HDV and HBV may cause fulminant hepatitis. MLID85155884 50 Persons with hemophilia who are chronic HBV carriers may also have antibodies to HDV, indicating exposure to HDV through factor concentrate. Even with virucidal methods used currently to prepare factor concentrate, HBV infections have been reported. Thus, all newly diagnosed persons with hemophilia should receive HBV vaccination. In a newly diagnosed infant, the series of three innoculations is started at birth. HCV has been a common infectious complication of factor infusion. (It was termed non-A, non-B hepatitis in the 1970s. MLID77202197 52) A serologic marker for HCV is available. MLID89222454 MLID89222455 53,54 Data suggest that E80% of persons with hemophilia infused before 1985 carry the marker, indicating past exposure to the virus. Approximately 90% of hemophiliacs have either persistently or intermittently elevated liver enzymes, which most likely represents the consequences of HCV exposure. 55 When liver biopsies are done on selected hemophilia patients with abnormal liver enzymes, 20%–30% have shown changes consistent with chronic active hepatitis or cirrhosis. MLID85253208 56 HCV has also been associated with the development of heptacellular carcinoma. MLID91314600 57 Thus, HCV may represent a long-term problem for persons with hemophilia. Outbreaks of hepatitis A related to factor concentrate have recently been reported from Europe. As a vaccine for hepatitis A becomes available, newly diagnosed persons with hemophilia should receive it. Human Immunodeficiency Virus HIV was introduced into the American blood supply in the 1970s. 58 By the late 1970s, factor concentrate was widely contaminated and by 1982, approximately 50% of persons with hemophilia were infected with HIV. 59 Currently, 70% of American hemophiliacs are HIV antibody positive. 60 Hemophilic patients, especially those infected after the age of 22 years and those who have been HIV seropositive for at least 7 years, have approximately 40% probability of developing symptomatic AIDS. MLID87239713 61 It is not clear why the age of acquisition of infection should influence outcome, but this has now been confirmed in a larger multicenter study. MLID90015005 62 Otherwise, when compared with other high-risk groups, the course of HIV-1 infection in hemophilia is very similar. As in other risk groups, low CD4 lymphocyte levels are strong predictors of which specific person will become symptomatic. Pneumocystis carinii pneumonia (Fig. 106-12) was the most common presenting AIDS-defining condition in HIV-seropositive hemophilia patients before the advent of prophylaxis. Other opportunistic infections, such as candida esophagitis and cryptococcal meningitis/septicemia, are reported in this subgroup. Kaposi sarcoma, however, is a very rare presenting condition in persons with hemophilia. Non-Hodgkin lymphoma can occur late in the disease at an incidence of 5.5%. MLID93214045 63 Currently, as with other HIV-seropositive patients, persons with hemophilia who have CD4 lymphocyte counts <400–500 cells/ml can be considered for prophylactic zidovudine; in those with a count of <200 cells/mm3, Pneumocystis prophylaxis should be added. The use of antiretroviral drugs that are hepatotoxic may be more problematic in persons with hemophilia since many have pre-existing liver disease secondary to HCV. Thus, liver chemistries should be monitored carefully, especially in persons on combination antiretroviral therapy. Virucidal Treatments of Concentrates There is now a triple barrier to viral transmission through factor concentrates: (1) self-exclusion for donors, (2) donor screening, and (3) viral inactivation procedures. Self-exclusion includes asking the plasma donor detailed questions concerning hepatitis, possible HIV exposure, and general health (to elicit nonspecific symptoms of HIV infection). Donor screening now includes HCV testing, as well as serologic testing for HIV-1 and HBV. Multiple methodologies for attenuating viruses during processing of factor concentrate have been devised. MLID89274385 64 They include heating the concentrate and the use of solvent/detergent combinations, which disrupt lipid-coated viruses such as HIV and some hepatitis viruses. MLID94054272 65,66 A third methodology for eliminating virus from concentrate involves affinity chromatography using a murine monoclonal antibody to either von Willebrand factor or factor VIII. MLID88264483 67 Cryoprecipitate is used as the starting material. A much higher purity factor VIII concentrate results (specific activity >3,000 U/mg protein before addition of albumin stabilizer). HIV titers are reduced significantly by this process. The product is either pasteurized in the final stage (Monoclate-P, Rorer) or is initially treated with tri-n-butyl phosphate (TNBP)/Triton X-100 (Hemophil M, Hyland). A summary of these methods and specific concentrates is given in Table 106-2 Table 106-2. Alloantigens in Factor Concentrate In addition to viral contamination of factor concentrates, it has been shown (beginning in the early 1980s) that intermediate purity concentrate itself may cause immune aberrations in hemophiliacs, perhaps secondary to the presence of multiple foreign proteins. MLID84244712 68 Factor concentrate in vitro may down-regulate Fc receptors on the macrophage MLID87158064 69 or may inhibit the mixed lymphocyte culture reaction MLID88264484 70 or interleukin-2 production by monocytes. MLID89194118 71 Since detailed immunologic studies were not done routinely on the hemophilia population before infection of patients with HIV, information regarding the effect of concentrate on patients is sporadic. In Scotland, in a group of HIV-seronegative hemophiliacs receiving locally produced factor concentrate not contaminated by HIV, approximately 50% demonstrated mildly decreased helper/suppressor T-lymphocyte ratios secondary to depressed CD4 cell levels. MLID84244712 68 These abnormalities appeared to be related to intensity of treatment. Others researchers have also shown that in a group of HIV-seronegative hemophiliacs, CD4 cell levels are mildly reduced when compared with normal controls. 72 Thus, frequent infusions of intermediate purity factor concentrates may lead to mild immune suppression. Additional Complications Other complications of treatment with concentrate include urticaria, temperature elevations, and very rarely anaphylaxis. Urticaria and bronchospasm are more commonly seen with infusions of cryoprecipitate. When cryoprecipitate is used, some centers order type-specific products. Factor concentrates may also contain measurable titers of isoagglutinins (anti-A or anti-B). When concentrate is administered in large amounts, such as postoperatively, to patients with A and B blood types, significant hemolysis may occur. MLID72211952 73 If the patient with this complication requires blood transfusions, type O should be given. Factor concentrate with low isoagglutinin titer can subsequently be obtained from specific manufacturers. Rarer complications of factor concentrate include a syndrome of primary pulmonary hypertension described in five patients with severe hemophilia A who used large amounts of concentrate. MLID88220869 74 The mechanism has not been elaborated but may be due to particulate matter or immune complexes being deposited in the lungs. A summary of infectious implications associated with therapy is given in Table 106-3 Table 106-3. Choice of Concentrate Selection of a factor concentrate depends on efficacy, safety concerning virally transmitted diseases, purity, and cost. Demonstrating that a concentrate is free of harmful viruses is difficult since reliable animal models, especially for HCV, are not available. Human clinical trials are necessary to prove that new factor concentrates are safe. To demonstrate whether a specific concentrate is free of HBV, HCV, or HIV, studies using previously nontransfused patients, primarily newly diagnosed hemophiliac infants, are undertaken. Serologic studies of antiviral antibodies and liver function may be positive if patients are exposed to specific viruses. Both HCV and HBV are difficult to inactivate. Heating the lyophilized concentrate at 60–68ºC for 30–72 hours does not inactivate HCV, although raising the temperature to 80ºC for 72 hours does eliminate HCV. MLID94054272 66 Heating the concentrate in solution (60ºC for 10 hours of "pasteurization") appears to kill HCV, although there are anecdotal reports that hepatitis B and C have still occurred after pasteurization methods were used. MLID87144520 MLID89013554 75,76 Solvent/detergent-treated concentrates appear to be free of HCV. MLID88287648 77 Of note is that solvent/detergent methods do not inactivate viruses without lipid envelopes. Thus, parvovirus, for example, may not be killed. In Ireland, Italy, Germany, and Belgium a recent outbreak of hepatitis A occurred in 84 hemophilia patients infused exclusively with solvent/detergent-inactivated concentrates purified by ion exchange chromatography. MLID92194930 78 HIV appears to be easily inactivated by any of the current viral inactivation procedures. In all viral safety trials in which HIV antibody status was studied, which include over 300 subjects, no seroconversions occurred. MLID89274385 64 However, 18 cases of HIV seroconversion have been reported with heat-treated factor VIII concentrate not associated with viral safety trials. 79 Most cases used concentrate that was dry heated at 60ºC, a methodology that is no longer used. "Dry heat" at high temperatures for longer periods of time (80º for 72 hours), vapor treatment, or heat treatment in solution ("pasteurization") appear to be efficacious in killing HIV. The solvent/detergent methods, as well as purification by affinity chromatography, also appear safe, vis-à-vis HIV infection. Purity of Factor VIII It is our opinion that for previously untreated and infrequently treated hemophiliacs and for others who are free of HIV infection, a concentrate that is pasteurized, treated with solvent/detergent, or immunoaffinity purified can be used. It should be virus free, but not necessarily highly purified. For HIV-seropositive patients, highly purified concentrates that are virally inactivated offer theoretical benefits. Highly Purified Concentrate Concentrates purified using affinity chromatography (Monoclate, Hemophil M) have a final specific factor VIII activity of approximately 3,000 U/mg protein. MLID88264483 67 The purity is significantly higher than previously available products, and most extraneous human proteins have been removed. These products are efficacious and appear to be free of hepatitis viruses and HIV. Since extraneous proteins such as immune complexes, aggregated immunoglobulins, and the killed viruses may be additionally suppressive to the immune system of a hemophiliac, concentrates containing only factor VIII and albumin may be less immunosuppressive. This would theoretically be beneficial not only to previously untransfused patients, but to HIV-seropositive patients as well. An early nonrandomized study of 14 HIV-1-seropositive patients compared the initial seven patients on high-purity factor VIII concentrate with seven patients on intermediate-purity concentrate (specific activity 1–3 U/mg protein); in the first group CD4 cell counts stabilized over the 3-year course of the study, while the second group had a decrease in CD4 counts. MLID89229428 80 There are now three randomized prospective trials comparing the effects of intermediate-purity versus high-purity concentrates on the immune system of HIV-1 seropositive hemophiliacs. 81 Two studies, using immune affinity-purified factor VIII, have shown independently that HIV-1-seropositive hemophilia patients have a slower decline of CD4 cell counts on the very high-purity concentrate versus the intermediate-purity concentrate. 82,83 In a third study using a less high-purity concentrate, no difference between the two groups could be seen. MLID92351359 84 All studies were performed with small numbers of patients. Recombinant Factor VIII Currently, there are three recombinant factor VIII products approved for clinical use, Recombinate (Baxter), Kogenate (Miles), and Bioclate (Armour). The final products are highly purified factor VIII, with a specific activity of approximately 7,000 U/mg protein prior to the addition of human albumin. Recombinant factor VIII has been shown to be safe and as efficacious as plasma-derived factor VIII concentrate, with similar recovery and half-life. MLID91061845 13,14,85 Major surgery has been performed using the concentrate, with excellent hemostasis. Two ongoing clinical trials are evaluating inhibitor development in persons with hemophilia treated with recombinant factor VIII. The patients in these trials are those most at risk of inhibitor development: persons who have previously been untransfused, mostly infants. To date, 16 of 64 patients developed an inhibitor after a median of 9 exposure days. MLID93133254 86 Although this prevalence is high, in 9 of the 16 patients, inhibitors remained at low titer. In another study, 17 of 69 previously untransfused patients given recombinate by infusion developed inhibitors. MLID94220686 87 It is not clear whether recombinant factor concentrate leads to increased inhibitor development or inhibitor development at an earlier time. Previous studies showing inhibitor prevalences of 10–15% were cross-sectional cohort, not prospective studies in a high-risk group. Also, the patients were not monitored as closely for inhibitor development. Thus, these studies are difficult to compare. Factor VIII: Recombinant Versus Plasma Derived Who should receive recombinant factor concentrate? There are many different opinions; ours is that this product should be reserved for those persons with hemophilia who are HIV-1 seronegative as well as HCV seronegative (i.e., mostly young children). The product is E$0.10/unit more expensive than the most expensive plasma-derived high-purity product. In this era of medical cost containment, price is a large issue. In addition, the possibility of inhibitor development must be discussed with the family or patient who will receive recombinant factor VIII. Cure of Hemophilia A Liver transplantation currently offers a cure for hemophilia, albeit an impractical one. Persons with hemophilia A have undergone liver transplantation because of end-stage liver disease caused by HBV or HCV. In one of the first series (four patients with hemophilia A), three survived the initial surgery and then normalized their factor VIII levels, requiring no further replacement. MLID87214539 88 Additional patients with hemophilia A have since undergone liver transplantation; of those who survived, factor VIII levels have been normalized. In the future, gene replacement therapy may offer a hope of cure to all persons with hemophilia. Genetic Counseling and Prenatal Diagnosis The cloning of the factor VIII gene and the determination of its molecular structure allow detection of carriers and accurate prenatal diagnosis. Female relatives of individuals with hemophilia may be carriers of the abnormal gene, and therefore may be at risk of having children with hemophilia. Women who are considered obligate carriers are daughters of individuals with hemophilia and women who have one son and another relative with hemophilia. These women have a 50% risk of each newborn son having hemophilia. Women who have two sons with hemophilia are obligate heterozygotes. However, a small proportion of these women may be either somatic mosaics or germline mosaics. MLID90368079 89 Reproductive risk in these circumstances is difficult to assess and depends on the proportion of ova carrying the abnormal gene. Other female relatives are considered possible carriers of the gene for hemophilia. This would include women who have one son with hemophilia and no other affected relatives. In these isolated cases, the hemophilia may result from (1) transmission through asymptomatic females, (2) a new mutation in the mother, (3) a new mutation in the individual with hemophilia (a true de novo mutation), or (4) as a result of somatic or germline mosaicism in the mother. MLID89126635 90 The probability for carriership for a mother of an isolated case is estimated to be 0.85. MLID93271395 91 Carrier Detection Potential hemophilia A carriers should be offered genetic testing for determination of carrier status. Current methods of carrier detection include standard phenotypic clotting assays, as well as more accurate genotypic analysis. Probability of carriership should first be determined from pedigree data. Information anterior to the proband is used to determine genetic risk. This figure can be modified by Bayesean analysis if the individual has any sons without hemophilia. Phenotypic assessment of carrier status for hemophilia A is determined by specific assays for factor VIII activity, factor VIII antigen, and von Willebrand factor antigen. In general, women who are carriers of hemophilia A have approximately 50% of the normal level of factor VIII. These values may be affected by various physiologic conditions or medications, or both. Of particular note are the effect of pregnancy (especially after the 22nd week of gestation) and estrogen-containing drugs such as birth control pills, which elevate factor VIII levels. MLID83049576 MLID75189904 92,93 The age of the individual being tested and the ABO blood type must also be noted; blood type O is associated with decreased factor VIII levels. MLID86216617 94 Factor VIII concentrations are also influenced by X-chromosome inactivation (the Lyon hypothesis), which may cause false-negative results. 95 Laboratory data are used to determine probability of carriership. The laboratory values can be combined with the pedigree data to give a final probability. For determination of odds ratios favoring carriership in hemophilia A, bivariate linear discriminant analysis using factor VIII, von Willebrand antigen, age, and ABO blood type is recommended. Determination of carrier status for hemophilia A may also be performed using molecular diagnostic methods. The factor VIII gene is large (see Ch. 105), and the mutations identified to date are heterogeneous. Thus, direct mutational analysis is not available for routine screening. Most genotypic testing employs an indirect marker, restriction fragment length polymorphism (RFLP). Which marker to use for polymorphism analysis is determined by the mutation's location (intragenic versus extragenic), the degree of heterozygosity, and the ethnic origin of the family. Southern blot analysis and polymerase chain reaction are the techniques used for identification of these polymorphisms. For hemophilia A, >95% of females are informative, with the following intragenic markers: the intron 13 CA repeat, the intron 22 CA repeat, BclI, and XbaI. The BglI and the intron 7 polymorphism may be informative in other families. Indirect testing using intragenic markers is >99% accurate. However, there are limitations. For the study to be informative, the marker must be heterozygous. Heterozygosity differs significantly in various ethnic groups, and this must be accounted for when considering which polymorphisms to use in a particular family. Blood samples from several key family members are required for genotypic testing. Blood from an affected male is required. Since many older hemophiliacs are infected with HIV-1 and thus have shortened survival, blood sampling to obtain DNA that can then be frozen for future use in genetic analysis should be encouraged. All family members must agree to genotypic testing, and all family relationships reported must be correct. In some families linked extragenic markers may be informative, but accuracy of the results is decreased due to possible genetic recombination. When sporadic cases of hemophilia occur in a family, polymorphism testing can only be used to exclude transmission of the mutation because the mutation is not identified by these methods, and therefore its origin is not known. In these families, direct mutation analysis can be considered. Direct screening methods for mutations in these large genes have been developed. Three screening methods are utilized for hemophilia A: denaturing gradient gel electrophoresis, single-stranded conformational polymorphism analysis, and direct sequencing of amplified DNA. MLID90169988 MLID89323457 MLID90152691 MLID94100976 96–100 Prenatal Diagnosis Several techniques are available for the prenatal diagnosis of hemophilia. The basic strategy is to determine fetal sex first, and then to determine whether the fetus has the gene for hemophilia. Fetal sex can be determined by ultrasonography and fetal chromosome analysis. Experienced sonographers can determine fetal sex reliably at 16–20 weeks of gestation. Fetal sex, however, is more accurately determined by examination of the fetal chromosomes. Chromosomes can be obtained by amniocentesis or chorionic villus sampling (CVS). Amniocentesis is performed at 15–20 weeks' gestation or at 12–14 weeks' gestation ("early amniocentesis"—risk approximately 1%). MLID92373696 101,102 CVS, by either the transcervical or the transabdominal route, is done at 9–12 weeks' gestation. MLID89143658 MLID92350194 103,104 In general, whether amniocentesis or a CVS procedure is chosen depends on patient preferences regarding timing of the procedure, risk to the fetus, and reliability of the results. Reports suggesting an association between CVS and limb reduction defects have raised questions about the risks of this procedure. These risks can be lessened if the CVS procedure is performed after 10 weeks' gestation, and if placental trauma is minimized. The risk of loss of pregnancy may be somewhat higher for CVS compared with amniocentesis. The diagnosis of hemophilia in a fetus can be accomplished by genotypic analysis of fetal DNA or by phenotypic analysis of fetal blood. Fetal DNA can be extracted from both fetal amniocytes and chorionic villi and analyzed by RFLP of the DNA, as above. If prenatal diagnosis is not possible by DNA analysis, then fetal blood sampling can be done by a cordocentesis. This technique is done from 20–21 weeks' gestation and involves the ultrasound-guided puncture of an umbilical cord vessel. MLID86047995 105 The fetal blood is analyzed by phenotypic clotting assays. Preimplantation diagnosis after in vitro fertilization has recently been proposed as a method of prenatal testing. Amplification of informative regions of the factor VIII gene by polymerase chain reaction from a single cell has been reported. All women who undergo carrier testing or prenatal diagnosis, or both, for hemophilia should have genetic counseling, preferably before choices regarding carrier testing and prenatal diagnosis must be made. The aims of counseling in these cases are to provide information about genetic risk, determination of carrier status, and prenatal diagnosis as well as to provide psychological and emotional support during the processes.